Mechanism of Abietadiene Synthase Catalysis: Stereochemistry and

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Mechanism of Abietadiene Synthase Catalysis: Stereochemistry and Stabilization of the Cryptic Pimarenyl Carbocation Intermediates Matthew M. Ravn,† Reuben J. Peters,‡,§ Robert M. Coates,*,† and Rodney Croteau*,‡ Contribution from the Department of Chemistry, UniVersity of Illinois, 600 S. Mathews AVenue, Urbana, Illinois 61801, and Institute of Biological Chemistry, Washington State UniVersity, Pullman, Washington 99164-6340 Received December 11, 2001. Revised Manuscript Received March 20, 2002

Abstract: Abietadiene synthase (AS) catalyzes the complex cyclization-rearrangement of (E,E,E)geranylgeranyl diphosphate (8, GGPP) to a mixture of abietadiene (1a), double bond isomers 2a-4a and pimaradienes 5a-7a as a key step in the biosynthesis of the abietane resin acid constituents (1b-4b) of conifer oleoresin. The reaction proceeds at two active sites by way of the intermediate, copalyl diphosphate (9). In the second site, a putative tricyclic pimaradiene or pimarenyl(+) carbocation intermediate of undefined C13 stereochemistry and annular double bond position is formed. Three 8-oxy-17-nor analogues of 9 (17 and 19a,b) and three isomeric 15,16-bisnorpimarenyl-N-methylamines (26a-c) were synthesized and evaluated as alternative substrates and/or inhibitors for recombinant AS from grand fir. The stereospecific cyclization of 8R-hydroxy-17-nor CPP (19a) to 17-normanoyl oxide (20a) and the higher inhibitory potency of the norpimarenylamine 26a (Ki ) 0.1 nM) both suggest pimarenyl intermediates having the 13β methyl configuration and 8,14-double bond corresponding to sandaracopimaradiene (5a). The 2000-fold stimulation of inhibition by 26a in the presence of inorganic pyrophosphate indicates an important role for carbocation/ OPP anion stabilization of the secondary sandaracopimaren-15-yl(+) ion. The failure of 8β-hydroxy-17nor CPP (19b) to undergo enzymatic cyclization was taken as evidence that 9 is bound with a “coplanar” side chain conformation and that the SN′ cyclization occurs on the 17R face. The routing of the sandarcopimara-15-en-8-yl carbocation toward various diterpenes in biogenetic schemes is attributed to differing conformations of ring C and/or orientations of the C13 vinyl group in the active sites of the corresponding diterpene cyclases.

Introduction

The resin acids are bicyclic and tricyclic diterpene carboxylic acids that occur in conifer oleoresin (pitch).1 The most abundant of these natural products belong to the abietane family of perhydrophenanthrene diterpenes characterized by an isopropyl substituent at C13, a conjugated diene in the B and/or C rings, and a C18 equatorial carboxyl group, i.e., abietic, levopimaric, neoabietic, and palustric acids (Figure 1, 1b-4b).1,2 The principal resin acids are usually accompanied by smaller proportions of the pimarane (13R methyl) and isopimarane (13β methyl) isomers differentiated by a quaternary carbon at C13 bearing methyl and vinyl substituents and an annular double bond at the 7,8 or 8,14 positions, as exemplified by sandaracopimaric, pimaric, and isopimaric acids (5b-7b). The struc* Corresponding authors. R.M.C.: tel. (217) 333-4280; fax (217) 2448024; E-mail [email protected]. R.C.: tel. (509)-335-1790; fax (509) 335-7643; E-mail [email protected]. † University of Illinois. ‡ Washington State University. § Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. (1) Soltes, J.; Zinkel, D. F. In NaVal Stores Production, Chemistry, and Utilization; Zinkel, D. F., Russell, J., Eds.; Pulp Chemicals Association: New York, 1989; Chapter 9. (2) Dev, S.; Misra, R. CRC Handbook of Terpenoids: Diterpenoids; Dev, S., Ed.; CRC Press: Boca Raton, FL, 1986; Vol. III. 6998

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tures of the tricyclic resin acids were determined by classical chemical degradations during the 1880s through 1950,3 and total and partial syntheses of abietic acid (1b) as well as the parent hydrocarbons, sandaracopimaradiene, pimaradiene, and isopimaradiene (5a-7a), have been reported.4 Oleoresin is secreted by conifers in response to insect attack and physical injuries, and this complex mixture of terpenoids serves to protect and seal the injury by oxidative cross-linking of the nonvolatile diterpene components after evaporation of the volatile monoterpene constituents.1,5 Processed gum rosin, composed primarily of abietic acid, is an abundant chemical commodity having numerous commercial applications.1,6 The readily isolated abietic and levopimaric acids7 have been used (3) (a) Simonsen, J.; Barton, D. H. R. The Sesquiterpenes, Diterpenes, and their DeriVatiVes, Vol. III of The Terpenes; Cambridge University Press: London, 1961; pp 374-458. (b) de Mayo, P. The Higher Terpenoids; Interscience: New York, 1959; pp 1-24. (c) Fieser, L. F.; Fieser, M. Natural Products Related to Phenanthrene; Reinhold: New York, 1949; Chapter II. (d) Nakanishi, K.; et al. In Natural Products Chemistry; Nakanishi, K., Goto, T., Itoˆ, S., Natori, S., Nozoe, S., Eds.; Kodanska Ltd.: Tokyo; Academic Press: New York, 1974; Vol. 1, Chapter 4. (4) Goldsmith, D. J. In The Total Synthesis of Natural Products; ApSimon, J., Ed.; Wiley: New York, 1992; Vol. 8, Chapter 1, pp 24-36. (5) Phillips, M. A.; Croteau, R. B. Trends Plant Sci. 1999, 4, 184-190. (6) (a) Reference 1, Chapters 16-21. (b) McCoy, M. Chem. Eng. News 2000, March 27, 13-15. 10.1021/ja017734b CCC: $22.00 © 2002 American Chemical Society

Mechanism of Abietadiene Synthase Catalysis

ARTICLES Scheme 1

Figure 1. Structures of the common abietane and pimarane types of diterpene hydrocarbons and the corresponding resins acid constituents: abietic, levopimaric, neoabietic, palustric, sandaraopimaric, isopimaric, and pimaric acids (1b-7b, respectively). The numbers in parenthesis are the relative amounts of each diterpene produced by abietadiene synthase and present as the acids in fresh oleoresin.

extensively as starting materials for synthesis of other diterpenes and various carbocyclic natural products and for chemical investigations.3,4,8 Abietadiene synthase9 is a multiproduct cyclase that generates a mixture of abietadiene (1a, 31%), levopimaradiene (2a, 34%), neoabietadiene (3a, 28%), palustradiene (4a, 2%), sandaracopimaradiene (5a, 2%), and pimaradiene (6a, 3%),10 a composition similar to that of the corresponding resin acids in fresh grand fir oleoresin.11 Abietadiene oxidases that convert the abietadiene to abietic acid have been isolated from grand fir and lodgepole pine.12 Recombinant abietadiene synthase (rAS) is an operationally soluble, monomeric 84-kDa protein sharing general characteristics with other terpene cyclases of plant origin, including the conserved DDXXD motif in the C-terminal domain together and a functionally important DXDD motif in the N-terminal domain.13 The structural relationshships among the resin acids led Ruzı´cka, Eschenmoser, and Heusser to propose their biogenetic origin by the sequence geranylgeraniol f labdanes (manool) f pimaradienes (pimaric acid) f abietadienes (abietic acid).14 The acid-catalyzed rearrangement of pimaric and isopimaric acids (6b and 7b) to abietic acid afforded relevant chemical precedent for the last step in this prescient biogenetic scheme.15 The now well-established biosynthesis of ent-kaurene (7) (a) Harris, G. C.; Sanderson, T. F. Organic Syntheses; Wiley: New York, 1963; Collect. Vol. IV, p 1. (b) Lloyd, W. D.; Hedrick, G. W. Organic Syntheses; Wiley: New York, 1973; Collect. Vol. V, pp 699-702. (8) (a) Arno, M.; Gonzalez, M. A.; Marin, M. L.; Zaragoza, R. J. J. Org. Chem. 2000, 65, 840-846. (b) Ayer, W. A.; Talamas, F. X. Can. J. Chem. 1988, 66, 1675-1685. (9) (a) LaFever, R. E.; Stofer Vogel, B.; Croteau, R. Arch. Biochem. Biophys. 1994, 313, 139-149. (b) Stofer Vogel, B.; Wildung, M. R.; Vogel, G.; Croteau, R. J. Biol. Chem. 1996, 271, 23262-23268. (10) (a) Peters, R. J.; Flory, J. E.; Jetter, R.; Ravn, M. M.; Lee, H.-J.; Coates, R. M.; Croteau, R. Biochemistry 2000, 39, 15592. (b) Lee, H.-J.; Ravn, M. M.; Coates, R. M. Tetrahedron 2001, 57, 6155-6167. (11) Lewinsohn, E.; Savage, T. J.; Gizen, M.; Croteau, R. Phytochem. Anal. 1993, 4, 220-225. (12) Funk, C.; Croteau, R. Arch. Biochem. Biophys. 1994, 308, 258-266. (13) Peters, R. J.; Ravn, M. M.; Coates, R. M.; Croteau, R. B. J. Am. Chem. Soc. 2001, 123, 8974-8978. (14) Ruzı´cka, L.; Eschenmoser, A.; Heusser, H. Experentia 1953, 9, 357-367.

via ent-copalyl diphosphate16,17 provided analogy for a proposed mechanism for the AS reaction by (1) proton-initiated polyene cyclization of (E,E,E)-geranylgeranyl diphosphate (GGPP, 8) to copalyl diphosphate (CPP, 9), (2) diphosphate ionization and ring closure to an indeterminate pimarenyl(+) or pimaradiene intermediate (10+, 5a, 6a, or 7a), and (3) proton-induced rearrangement to abietadiene (1a) (Scheme 1).9 CPP proved to be a competent substrate for rAS, undergoing conversion to the same mixture of abietadiene and pimaradiene isomers 1a-6a.10a Steady-state kinetic analysis indicated that the rate-limiting step occurs after formation of CPP because the calculated kcat is 2.2 s-1 for either GGPP or CPP, although substrate inhibition (Ki ) 5 µM) prevents direct measurement of the rate from GGPP. The catalytic efficiency for CPP (KM ) 0.4 µM, kcat/KM ) 5.5 × 106 s-1 M-1) proved to be 8 times greater than that of GGPP (KM ) 3 µM, kcat/KM ) 7.3 × 105 s-1 M-1) owing to the tighter binding of CPP.10a,13 The presence of two active sites, one for the conversion of GGPP to CPP and the other for the cyclizationrearrangement of CPP to the abietadiene mixture, has been established by selective inhibition of the first reaction by 15aza-14,15-dihydroGGPP while retaining the second catalytic activity, as well as by site-directed mutagenesis.13 rAS catalyzed cyclizations of the labeled substrates [19,19,19-2H3]GGPP, (17E)-[17-2H1]CPP, and (15R, 17E)[15-2H1,17-3H1]CPP revealed that the conversion of CPP to abietadiene occurs with stereospecific intramolecular transfer of the C17 pro-E proton to the C16 si face of the vinyl group in the pimaradiene or pimaren-8-yl ion intermediate. This protonation is followed by syn methyl migration to the same vinyl group face (15-si), thus creating the characteristic isopropyl group of the abietane products.18 However, the C13 stereochemistry (R-CH3 or β-CH3) and the annular double bond position (∆-7,8 or ∆-8,14) of the pimarenyl intermediate 11+ remained uncertain because none of the four plausible pima(15) Wenkert, E.; Chamberlin, J. W. J. Am. Chem. Soc. 1959, 81, 688-693. (16) Schecter, I.; West, C. A. J. Biol. Chem. 1969, 244, 3200-3209. (17) MacMillan, J.; Beale, M. H. In Isoprenoids Including Carotenoids and Steroids; Cane, D. E., Ed.; Vol. 2 in Comprehensive Natural Products Chemistry; Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.; Elsevier: Oxford, U.K., 1999; Chapter 8. (18) (a) Ravn, M. M.; Coates, R. M.; Jetter, R.; Croteau, R. B. Chem. Commun. 1998, 21-22. (b) Ravn, M. M.; Coates, R. M.; Flory, J. E.; Peters, R. J.; Croteau, R. Org. Lett. 2000, 2, 573-576. J. AM. CHEM. SOC.

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radiene isomers are converted to abietadiene by AS.9,10a The formation of such enzymatically enclosed intermediates in terpene synthase-catalyzed cyclizations (resulting in the failure of exogenous intermediates to participate in the reaction) is a now a well-precedented phenomenon;17,19 some specific examples are cineole, pentalenene, aristolochene, epi-aristolochene, vetispiradiene, and taxadiene synthases.20 In this paper we report syntheses of three 8-oxy-17-nor analogues of copalyl diphosphate (9) and three isomeric pimarenylammonium salts that mimic the putative pimaren-15yl carbocation 11+. The AS-catalyzed reactions and/or the inhibitory properties of these and related compounds toward this unusual diterpene synthase shed light on the stereochemistry of the cyclization and rearrangements which generate the abietane structure and on the nature and stabilization of the carbocation intermediates in the mechanism.21

Scheme 2

8-Oxy 17-Norcopalyl Diphosphate Analogues

of the 8,17 and 13,14 regioisomeric diols. Reduction of the mixture of methyl esters (LiAlH4, Et2O, 0 °C, 10 h) followed by oxidative cleavage (NaIO4, 6:1 acetone-water, 24 h)18b,29 gave 8-oxo-17-norcopalol (16, 60% for two steps) and the chromatographically separable methyl ketone arising from the 13,14-diol. The epimeric 8R- and 8β-hydroxy norcopalol analogues were obtained by stereoselective reductions of 16 under dissolving metal conditions (Li/NH3, EtOH, Et2O, -33 °C, 83%),30 or with metal hydride (NaBH4, MeOH, 74%), to give diols 18a,b, respectively, as crystalline solids. The equatorial and axial positions of the hydroxyl groups in the isomers were established from the 1H NMR couplings of the H8 protons: 18a, δH 3.41 (td, J ) 10.6, 4.9 Hz); 18b, δH 3.95(q, J ) 2.6 Hz). Despite the possibility of SN′ cyclization with the proximal carbonyl group, phosphorylation of keto alcohol 16 proceeded smoothly under standard conditions by conversion to the allylic chloride (MsCl, LiCl, s-collidine, DMF, 0 °C)31 followed by displacement with tris(tetrabutylammonium) diphosphate ((Bu4N)3HP2O7‚3H2O, CH3CN, 78%)32 which afforded 8-oxo-17-norcopalyl diphosphate (17) as the pure ammonium salt after ionexchange and cellulose chromatography. In contrast, attempted conversions of diols 18a,b to the corresponding allylic chlorides by the same procedure resulted only in mixtures of cyclization and addition products. Instead, activation by regioselective phosphorylations (ClPO(OEt)2, py, CH2Cl2, 0 °C) followed by slow displacement with tris(tetrabutylammonium) diphosphate at high concentration (CH3CN, (NBu4)3HP2O7‚3H2O, 1 M, 4 d, 3 Å sieves, 68-69% for two steps)29 provided the primary allylic diphosphates 19a,b as NH4+ salts after ion-exchange and either preparative reverse-phase HPLC or cellulose chromatography. Chemical cyclizations of the nor-hydroxy analogues 18a,b were carried out to obtain independently prepared samples of the cyclic ethers and to establish their C13 configurations. Acid-catalyzed cyclization of equatorial diol 18a (p-TsOH‚H2O,

Both chemical and biochemical approaches have been utilized to identify transient intermediates in terpene cyclase reactions. The occurrence of an enzymatically enclosed germacrene A intermediate in sesquiterpene synthase-catalyzed cyclizations was verified by site-directed mutagenesis of an active site residue22a and by use of 6,7-dihydrofarnesyl diphosphate22b that is incapable of undergoing the full cyclization sequence. Support for putative carbocation intermediates in the cyclizationrearrangement cascade effected by oxidosqualene-lanosterol cyclase has been obtained by use of oxa, dehydro, methylidene, and other substrate analogues.23,24 We considered that 8-oxo and 8-hydroxy 17-nor analogues (Scheme 2; 17, 19a,b) of the bicyclic intermediate 9 might serve to reveal the cryptic C13 configuration of the pimarenyl intermediates and the stereochemistry of the SN′ cyclization which takes place at the second AS catalytic site. The readily available labdane diterpene manool25 was converted to (13E)-methyl copalate (13) by oxidative rearrangement with pyridinium chlorochromate (CH2Cl2, 80%)26 to a 2:1 mixture of (13E)- and (13Z)-copalals, followed by oxidation with MnO2-NaCN in methanol (93%).18b,27 Catalytic osmylation of 13 (OsO4, NMO, acetone, 3 h, 78%)28 provided a 3:1 mixture (19) (a) Wise, M. L.; Croteau, R. In ref 17, Chapter 5. (b) Cane, D. E. In ref 17, Chapter 6. (20) (a) Cineole synthase: Croteau, R.; Alonso, W. R.; Koepp, A. E.; Johnson, M. A. Arch. Biochem. Biophys. 1994, 309, 184-192. (b) Pentalenene synthase: Lesburg, C. A.; Zhai, G.; Cane, D. E.; Christianson, D. W. Science 1997, 277, 1820-1824. Cane, D. E.; Oliver, J. S.; Harrison, P. H. M.; Abell, C.; Hubbard, B. R.; Kane, C. T.; Lattman, R. J. Am. Chem. Soc. 1990, 112, 4513-4524. (c) Aristolochene synthase: Cane, D. E.; Prabhakaran, P. C.; Salaski, E. J.; Harrison, P. H. M.; Noguchi, H.; Rawlings, B. J. J. Am. Chem. Soc. 1989, 111, 8914-8916. (d) Epiaristolochene and vetispiradiene synthases: Starks, C. M.; Back, K.; Chappell, J.; Noel, J. P. Science 1997, 277, 1815-1820. Mathis, J. R.; Back, K.; Starks, C.; Noel, J.; Poulter, C. D.; Chappell, J. Biochemistry 1997, 36, 8340-8348. (e) Taxadiene synthase: Lin, X.; Hezari, M.; Koepp, A. E.; Floss, H. G.; Croteau, R. Biochemistry 1996, 35, 2968-2977. (21) Some aspects of this work were published in a preliminary communication.18b (22) (a) Rising, K. A.; Starks, C. M.; Noel, J. P.; Chappell, J. J. Am. Chem. Soc. 2000, 122, 1861-1866. (b) Cane, D. E.; Tzantrizos, Y. S. J. Am. Chem. Soc. 1996, 118, 10037-10040. (23) Abe, I.; Prestwich, G. D. In ref 17, Chapter 10. (24) (a) Abe, I.; Rohmer, M.; Prestwich, G. D. Chem. ReV. 1993, 93, 21892206. (b) Abe, I.; Tomesch, J. C.; Wattanasin, S.; Prestwich, G. D. Nat. Prod. Rep. 1994, 11, 279-302. (25) Manool was isolated from heartwood extract of Halocarpus biforme obtained from Westchem Industries Ltd., PO Box 708, Dunedin, NZ, with assistance from Prof. Brian Robinson, Chemistry Department, University of Otago. (26) Sundararaman, P.; Herz, W. J. Org. Chem. 1977, 42, 813-819. (27) Corey, E. J.; Gilman, N. W.; Ganem, B. E. J. Am. Chem. Soc. 1968, 90, 5616-5617. 7000 J. AM. CHEM. SOC.

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(28) VanRheenan, V.; Cha, D. Y.; Hartley, W. M. Organic Syntheses; Wiley: New York, 1988; Collect. Vol. VI, pp 342-348. (29) Ravn, M. M.; Jin, Q.; Coates, R. M. Eur. J. Org. Chem. 2000, 14011410. (30) Huffman, J. W.; Desai, R. C.; LaPrade, J. E. J. Org. Chem. 1983, 48, 14741479. (31) Meyers, A. I.; Collington, E. W. J. Org. Chem. 1971, 36, 3044-3045. (32) (a) Davisson, V. J.; Woodside, A. B.; Neal, T. R.; Stremler, K. E.; Muehlbacher, M.; Poulter, C. D. J. Org. Chem. 1986, 51, 4768-4779. (b) Woodside, A. B.; Huang, Z.; Poulter, C. D. Organic Syntheses; Wiley: New York, 1993; Collect. Vol. VIII, pp 616-620.

Mechanism of Abietadiene Synthase Catalysis Scheme 3

Scheme 4

ARTICLES

222 nm).34 The structure of the cyclic ether product was confirmed by GC comparisons with the 20a + 20b mixture and by 1H NMR spectra and NOE determinations on isomerically pure 20a from the enzymatic reaction. AS-catalyzed cyclization of (15R)-[15-2H1]19a29 afforded (16E)-[16-2H1]normanoyl oxide (20a-d1) bearing label in the trans position of the vinyl group judging from its 1H NMR spectrum: δH 5.37 (d, J ) 17.6 Hz), 6.04 (d, J ) 17.3 Hz). In contrast, incubations of the 8β-hydroxy isomer 19b with rAS under the same conditions gave no significant amounts of extractable products (conversions e 0.2% by GC), despite evidence cited below that the analogue is a strong competitive inhibitor of CPP, exhibiting Ki ) 0.2 µM, and thus clearly binding at the active site. Syntheses of Norpimarenylamine Inhibitors

Et2O, 1.5 h, room temperature)33 gave a 1.5:1 mixture (76%) of inseparable cyclic ethers (13R)-20a and (13S)-20b (Scheme 3). NOE experiments with irradiation of both overlapping isomeric H8 methine protons (δH 3.52 and 3.54 for major and minor isomers, respectively) showed strong correlations to the C17 (δH 1.21, 6.0%) and C20 (δH 0.74, 4.3%) methyls for the major isomer and to the C20 (δH 0.74, 4.3%) methyl and H15 (δH 5.79, 4.8%) vinyl proton for the minor isomer. It follows that the major isomer possesses an axial 13β methyl group (13R) and the minor isomer has an equatorial 13R methyl (13S). The 13β methyl isomer 20a proved to be the sole product of the AS-catalyzed cyclization of 8R-hydroxy-17-nor diphosphate, 19a. A somewhat slower cyclization of axial diol 18b under similar conditions (30 °C with TLC monitoring) gave a 3:1 mixture (47%) of cyclic ethers (13R)-21a and (13S)-21b and a 5:2.5:1 mixture (41%) of (12E)-12,14, (12Z)-12,14, and 13(16),14dienols, respectively, arising from competing 1,4-proton eliminations. NOE experiments on the mixture of cyclic ethers with irradiation of both overlapping isomeric H8 methine protons (δH 3.7-3.8) showed strong correlations to the H15 (δH 5.76, 10%) vinyl hydrogen for the major isomer (21a) and to the H17 methyl (δH 1.21, 2.6%) for the minor isomer (21b). The slower rate and the lower yield of the cyclic ether products formed from axial diol 18b are no doubt a response to steric interactions of the axial substiuents on incipient cis-fused C-ring with the C10 angular methyl group. Incubation of 8-oxo and 8R-hydroxy-17-nor analogues 17 and 19a with rAS resulted in regiospecific elimination of the former to 17-norlabda-13 (16),14-dien-8-one (22, 30% conversion by GC) and stereospecific SN′ cyclization of the latter to 17normanoyl oxide (13R, 20a, 46% conversion by GC), as the principal products according to GC analyses and NMR spectra (Scheme 4). Dienone 22 was identified by coupling patterns and integrations for the vinyl hydrogens in its 1H NMR spectra as well as its UV spectrum (λmax 226 nm, ethanol) which is similar to that predicted for a 2-alkyl-1,3-butadiene (calcd λmax

The norpimarenylammonium salts 26a-c are nitrogen mimics of three of the four possible secondary pimarenyl ions symbolized by intermediate 11+ shown in Scheme 1. Aza analogues of hypothetical carbocations have often proven to be potent inhibitors of many terpene cyclases as well as tranferases, isomerases, and oxidases associated with isoprenoid biosynthesis.24,35 It therefore seemed reasonable to expect that the norpimarenylamine isomer having the C13 configuration and double bond position corresponding to the transient carbocation 11+ would be a more potent inhibitor than its isomers. The three norpimarenylamines were synthesized by Curtius rearrangements of the corresponding 16-norpimaren-15-oic acids (24a-c) and direct hydride reductions of the resulting isocyanates (Scheme 5). A 9:1 mixture of sandaracopimaric and isopimaric acids (5b + 7b) isolated from sandarac resin33 and pimaric acid (6b)36 were converted in 5 steps to the corresponding pimaradienes, 5a + 7a mixture and 6a, by known procedures.37 Regioselective dihydroxylation (catalytic OsO4, NMO, acetone, rt, 88%)28,38 of the 5a + 7a mixture followed by periodate cleavage (NaIO4, acetone-water, rt, 3 h, 90%)39 and hydride reduction (LiAlH4, ether) furnished the corresponding primary alcohols which proved separable by careful chromatography on silica gel (23a, 69%; 23b, 5%). Jones oxidations (CrO3, H2SO4, acetone) gave the known40 nor acid 24a (67%) and its double bond isomer, 24b (86%). Curtius rearrangement of 24a with diphenylphosphoryl azide41 (DPPA, Et3N, PhH, reflux, 50 min) provided the stable isocyanate 25a (86%) that was purified by rapid chromatography on silica gel. Reductions of 25a with LiAlH4 in ether (rt, 2h) and THF (reflux, 2h) (33) Edwards, O. E.; Mootoo, B. S. Can. J. Chem. 1969, 47, 1189-1194. (34) Scott, A. I. Interpretation of the UltraViolet Spectra of Natural Products; MacMillan: New York, 1964; pp 45-50. (35) (a) Oehlschlager, A. C.; Angus, R. H.; Pierce, A. M.; Pierce, H. D., Jr.; Srinivasan, R. Biochemistry 1984, 23, 3582-3589. (b) Reardon, J. E.; Abeles, R. H. Biochemistry 1986, 25, 5609-5616. (c) Muelbacher, M.; Poulter, C. D. Biochemistry 1988, 27, 7315-7328. (d) Rahier, A.; Taton, M.; Benveniste, P. Biochem. Soc. Trans. 1990, 18, 48-52. (e) McGeady, P.; Pyun, H. J.; Coates, R. M.; Croteau, R. Arch. Biochem. Biophys. 1992, 299, 63-72. (f) Steiger, A.; Pyun, H.-J.; Coates, R. M. J. Org. Chem. 1992, 57, 3444-3449. (36) A 10:1 mixture of sandaracopimaric and isopimaric acids (5a + 7a) was isolated according to the procedures of Edwards and Mootoo.33 It was found later that pure 5a could be isolated readily by crystallization of the ethanolamine salt. See Supporting Information for details. Pimaric and isopimaric acids were obtained as gifts from Dr. Duane Zinkel at the USDA Forest Products Labratory, Madison, WI. (37) (a) Coates, R. M.; Kang, H. Y. J. Org. Chem. 1987, 52, 2065-2074. (b) Chu, M.; Coates, R. M. J. Org. Chem. 1992, 57, 4590-4597. (38) For regioselective dihydroxylations of pimaradiene diterpenes, see: (a) Ireland, R. E.; Newbould, J. J. Org. Chem. 1963, 28, 23-26. (b) Edwards, O. E.; Howe, R. Can. J. Chem. 1959, 37, 760-774. J. AM. CHEM. SOC.

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Ravn et al. Table 1. Inhibition and Kinetic Data for Oxy-nor CPP Analogs (17, 19a,b), Norpimarenylamines (26a-c), and Truncated Acyclic Substrate Analogs (27, 28)a

Scheme 5

compd

Kib (nM)

17 19a 19b 26a 26b 26c 27 28

120 ( 10 150 ( 30 200 ( 50 240 ( 10 1200 ( 200 11000 ( 700 8000 6d

+ 1 mM PPi Kic (nM)

kcat

product

∼0.03 ∼0.07

22 20a

0.13 ( 0.05d 2.8 ( 0.2d 400 ( 100d

a Inhibition was determined by utilizing [15-3H]-CPP as the substrate. Competitive inhibition. c Uncompetitive inhibition, presumably owing to the previously observed uncompetitive inhibition from divalent metal ion chelation occurring with 1 mM inorganic diphosphate.10a d Apparent Ki determined by fitting a tight binding inhibitor equation to a Dixon-type experiment.13 b

afforded the formamide or the N-methylamine, respectively, and the latter was isolated as its hydrochloride salt (26a, 56%). Similar procedures sufficed to convert 16-norisopimaren-15oic acid 24b and pimara-8(14),15-diene (6a) to 15,16-dihydro15-azaisopimaradiene and the corresponding azapimaradiene as HCl salts 26b,c in comparable yields. Inhibition and Kinetic Studies with Recombinant (-)-Abietadiene Synthase

Kinetic analyses of the synthetic substrate analogues and intermediate mimics with rAS were carried out to determine their relative rates and/or affinities for the active site (Table 1). Cyclization rates (kcat) for 17 and 19a were found to be ∼0.03 and ∼0.07 s-1 (krel 0.01 and 0.03 to those for GGPP; kcat ) 2.2 s-1). Products generated were quantified by GC using geranylgeraniol as an external standard and identified by preparative enzyme assays and comparisons with authentic samples obtained as discussed above. Interestingly, axial nor-hydroxy analogue 19b did not serve as a substrate for rAS, but instead acted as a competitive inhibitor of the cyclization of [15-3H]-(+)-CPP, exhibiting Ki ) 0.2 µM. Both 17 and 19a also served as effective inhibitors when added immediately prior to radiolabeled substrate. It was expected that the norpimarenylamine isomer corresponding to the cryptic pimarenyl intermediate 11+ would display the highest affinity for rAS and that there might exist synergy between exogenous diphosphate anion (mimicking the ionized diphosphate) and such carbocation mimetic analogues, as previously observed with other terpene synthases.42 Therefore, the three amines 26a-c were assayed as inhibitors against rAS with and without the addition of 1 mM inorganic diphosphate (PPi) (Table 1). As expected from the stereoselective cyclization of 19a to 20a, the isomers with the 13β methyl group, 26a,b, were significantly more potent inhibitors than 26c with the 13R methyl stereochemistry by 10-15-fold in the absence of inor7002 J. AM. CHEM. SOC.

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ganic pyrophosphate. The dramatic synergy exhibited by the amine inhibitors in the presence of the additive (30-2000-fold) indicates an enzyme-mediated ion pairing interaction between the pyrophosphate anion and the secondary pimarenyl carbocation. The inhibition kinetics of two acyclic substrate analogues, (E,E)-farnesyl diphosphate (FPP, 27) and farnesylmethyl-1′,1′bisphosphonate (FMBP, 28),43 as well as the cyclization rate of CPP selectively deuterated in the exocyclic methylene group were also studied to determine the effects of shortening the polyenyl chain, altering the orientation of the polar headgroup, and substituting the heavier isotope for the hydrogen that migrates to C16. The previously reported rAS-catalyzed conversion of FPP to the acylic tetraene (E)-β-farnesene most likely occurs by ionization and deprotonation at the CPP cyclization site where diphosphate heterolysis occurs.10a FPP proved to be a competitive inhibitor of rAS when added immediately prior to radiolabeled CPP, with Ki ) 8 µM. Interestingly, FMBP is a much better inhibitor of rAS with Ki ) 6 nM, possibly indicating a twist of the binding domains for the hydrocarbon and diphosphate moieties. Detailed kinetic analysis of the previously characterized (15R, 17E)-[15-3H1, 17-2H1]CPP18b revealed a small, but significant, deuterium isotope effect (KIE ) 1.4 ( 0.1) on the overall rate, indicating a small degree of C17-H bond-breaking in the transition state or a secondary KIE associated with the sp2 f sp3 rehybridization at C17 in the initiating SN′ cyclization step.

(39) Grigor’eva, N. Y.; Avrutov, I. M.; Semenovskii, A. V.; Odinokov, V. N.; Akhunova, V. R.; Tolstikov, G. A. IzV. Akad. Nauk., Ser. Khim. (Engl. Transl.) 1979, 28, 353-358. (40) (a) Ireland, R. E.; Schiess, P. W. J. Org. Chem. 1963, 28, 6-16. (b) Ireland, R. E.; Mander, L. N. J. Org. Chem. 1967, 32, 689-696. (41) Ninomiya, K.; Shioiri, T.; Yamada, S. Tetrahedron 1974, 30, 2151-2157. (42) (a) Cane, D. E.; Yang, G.; Coates, R. M.; Pyun, H.-J.; Hohn, T. M. J. Org. Chem. 1992, 57, 3454-3462. (b) Koohang, A.; Coates, R. M.; Owen, D.; Poulter, C. D. J. Org. Chem. 1999, 64, 6-7. (c) Croteau, R.; Wheeler, C. J.; Aksela, R.; Oehlschlager, A. C. J. Biol. Chem. 1986, 261, 7257-7263. (43) Ciosek, C. P., Jr.; Magnin, D. R.; Harrity, T. W.; Logan, J. V.; Dickson, J. K., Jr.; Gordon, E. M.; Hamilton, K. A.; Jolibois, K. G.; Kunselman, L. K.; Lawrence, R. M. J. Biol. Chem. 1993, 268, 24832-24837.

Mechanism of Abietadiene Synthase Catalysis

Discussion

The three 8-oxy analogues (17, 19a,b) of 9 proved to be active as alternative substrates and/or inhibitors of AS and provided useful insights concerning the stereospecifity of the SN′ cyclization step. The slow but stereospecific enzyme-catalyzed cyclization of the 8R-hydroxy analogue to 17-normanoyl oxide (19a f 20a, k19a/k9 ) 0.03) constitutes strong, indirect evidence that pimaren-8-yl intermediate 10+ in fact has the 13β methyl configuration corresponding to sandaracopimaradiene (5a),44 a minor byproduct formed with the normal substrate. The absence of any 14-oxa-abietene products that would arise by proton transfer from oxygen to C16 and methyl rearrangement is presumably attributable in part to the decreased Bronsted acidity of the oxonium ion intermediate compared to the pimarenyl carbocation 10+. It seems likely that the 8-oxo analogue 17 undergoes similar ring closure to a cyclic oxocarbenium ion that suffers ring cleavage and regiospecific β-elimination at the methyl group to form dienone 22. The AS-catalyzed cyclization of deuterium-labeled (15R)[15-2H1]-19a to (15E)-[16-2H1]-20a establishes an anti relationship of the 8R-OH nucleophile and the OPP- leaving group and, by inference, an anti SN′ cyclization for the normal copalyl PP intermediate 9. This conclusion conforms with similar outcomes reported for the biosynthesis of normal and enantiomeric diterpenes produced by fungi and higher plants.45,46 As a consequence, the initially formed pimaren-8-yl(+)/diphosphate(-) ion pair (10+/OPP-) would have the anion situated on the si,si face of the C13 vinyl group. Although the epimeric hydroxy analogue 19b clearly binds at the copalyl PP cyclization site, as shown by its strong inhibition (Ki19b/KMCPP ) 0.2 µM/0.35 µM ) 0.6), this compound with an axial 8β-OH as potential nucleophile fails to undergo enzyme-catalyzed cyclization or elimination. The acid-induced dehydration of the corresponding diol 18b to a mixture of epimeric cyclic ethers (21a + 21b, 47%, Scheme 3) indicates that the steric interactions arising from the cis B, C ring fusion should not be insurmountable. It seems reasonable to propose that copalyl PP and its 8-oxy-17-nor analogues are bound with the pentenyl diphosphate side chain oriented approximately coplanar to the A and B rings. In this conformation, C13-C17 bond formation would take place on the 17R face of the exocyclic methylene carbon (17 re) of 9, and the 8R-OH group of 19a would be suitably positioned for SN′ cyclization to normanoyl oxide. In contrast, the axial 8β-hydroxy group of 19b is too far from C13 to allow cyclization in the same “coplanar” side chain conformation, and the “bent” conformation alternative required for nucleophilic attack on C13 is, according to this interpretation, incompatible with the contours of the copalyl PP binding pocket. The relative potencies of the three pimarenylamine inhibitors 26a-c (Table 1) are consistent with the 13β methyl configu(44) This conclusion is supported by recently published results from mutational analysis of rAS. Mutant N765A proved unable to mediate the intramolecular proton transfer and produced unrearranged sandaracopimaradiene (5a) as the sole product. See: Peters, R. J.; Croteau, R. B. Proc. Acad. Sci. U.S.A. 2002, 99, 580-584. (45) (a) Drengler, K. A.; Coates, R. M. J. Chem. Soc., Chem. Commun. 1980, 856-857. (b) Drengler, K. A. Ph.D. Dissertation, University of Illinois, Urbana, IL, 1980. (46) (a) Cane, D. E.; Hasler, H.; Materna, J.; Cagnoli-Bellavita, N.; Ceccherelli, P.; Madruzza, G. F.; Polonski, J. J. Chem. Soc., Chem. Commun. 1981, 280-282. (b) Cane, D. E.; Murthy, P. P. N. J. Am. Chem. Soc. 1977, 99, 8327-8329.

ARTICLES Scheme 6

ration of the pimaren-15-yl ion intermediate inferred from the cyclization of 19a to 20a.44 Thus the Ki for 26a with the 13β methyl substituent is a factor of 46 lower than Ki for its epimer 26c with the opposite R methyl orientation at C13, and the Ki ratio decreases to 10-3 in the presence of 1 mM OPP-. The smaller but significant potency enhancement of 26a over its ring B double bond isomer 26b (5-fold in the absence of OPP- and 22-fold in the presence of 1 mM OPP-) indicates a ring C location for the double bond in the pimaren-15-yl(+) ion formed after proton transfer to C16, consistent with previous labeling results.18 The stimulating effect of inorganic pyrophosphate has been previously observed using aza and sulfonium analogues of tertiary carbocation intermediates in other terpene synthase reactions.35e,42 However, in these cases, the enhancements in affinity in the presence of OPP- were only about 1 order of magnitude, considerably less than the 3 orders of magnitude observed here for norpimarenylamine 26a. The results discussed above, together with those from prior deuterium and tritium labeling experiments,18 support the conformational mechanism illustrated in Scheme 6 for the conversion of 9 to the abietadiene isomers 1a-4a catalyzed by rAS.47 SN′ cyclization of 9 with the “coplanar” side chain orientation and bonding to the 17R face (17-re to 13-si) generates the pimar-15-en-8-yl(+) carbocation 10A+ with the 13β methyl group and ring C initially in a boat conformation. Ring inversion accompanied by ca. 150° rotation of the vinyl group gives rise to conformer 10B+. Migration of H14R (HC) in a stereoelectronically suitable axial orientation to the 16-si face of the vinyl group, either intramolecularly or indirectly by transfers to and from the AS active site interior by way of a transient sandaracopimaradiene intermediate (5a),44 leads to secondary pimar-8(14)-en-15-yl(+)/OPP- ion pair 11A+. Another -90° rotation about the C13-C15 bond sets the stage (47) (a) The possibility that the 14 f 16 proton transfer could occur with ring C in a boat conformation (10A+) appears unlikely for stereoelectronic reasons. Inspection of a Dreiding model of 10A+ shows that HC is aligned orthogonal to the carbocation orbital. (b) A reviewer suggested an alternative mechanism in which the CPP intermediate (9) undergoes enzyme-catalyzed allylic rearrangement to its tertiary isomer, epimanoyl diphosphate, a process well precedented in the cyclization mechanisms of monoterpene and sesquiterpene synthases.19 In this case, rotation of the vinyl group to a cisoid conformation could occur prior to cyclization of the epimanoyl PP intermediate. No evidence is presently available to exclude or support this somewhat more complicated mechanistic alternative. J. AM. CHEM. SOC.

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Ravn et al.

Scheme 7

It is intriguing to speculate upon the factors responsible for directing this common intermediate toward the structurally different diterpene products. The conformation of ring C, the orientation of the vinyl group, and the position of the diphosphate counterion probably all play critical roles. Maintenance of the boat conformation of 10A+ together with a 120° rotation of the vinyl group toward C8 allows C8-C16 bonding while avoiding transfer of the orthogonally disposed H14R. However, ring inversion 10A+ f 10B+, vinyl group rotation, and diphosphate positioning would prepare the ion to undergo 14-16 proton transfer and methyl rearrangement to the abietadiene family. If both of these outlets are prevented, e.g. by fixing the vinyl group in its initial extended orientation, the H9 to C8 hydride shift may take place to begin the backbone rearrangements to the 9β methyl diterpene family.

for migration of the 13β methyl to the 15-si face of the secondary ion to create the isopropyl group; subsequent proton eliminations from the periphery of the resulting allylic abietenyl ion would give rise to the observed 1a-4a isomer mixture. The suprafacial relationship of the C14-C16 proton transfer and the C13-C15 methyl migration evidently requires separate steps and the obligatory conversion of the more stable tertiary pimaren-8-yl ion 10B+ to the less stable secondary ion 11A+ as a discrete intermediate. The small kinetic isotope effect observed when deuterium is transferred is significantly less than the 2-5-fold change expected for a rate-determining primary isotope effect; thus the proton transfer (and the resulting tertiary to secondary carbocation transition) is at most only slightly ratelimiting. The 15 kcal/mol enthalpic difference between tertiary and secondary carbocations48 might be alleviated significantly by an associated decrease in the separation of the carbocation site and the diphosphate anion in the secondary ion. Measurements with molecular models indicate that the distance between the carbocation sites in 10B+//OPP- and 11A+/OPP- ion pairs is approximately 3.6-3.7 Å (distance between C8 and C15). The 5 × 10-4 decrease in Ki (∆∆G ∼ 4.5 kcal/mol) for primarenylamine inhibitor 26a in the presence of 1 mM diphosphate ion signifies an important role for the leaving group in the transition state composite and is likely a manifestation of ion pairing as a significant stabilizing influence in this seemingly energetically demanding isomerization. This ion pair interaction might also explain the observed suprafacial proton transfer and methyl migration (10+ f 12+ si face selectivity) previously discussed, as it would effectively shield one face of the olefin and enforce a stepwise mechanism. Substitution of the pyrophosphate by the methylbis(phosphonate) group dramatically increases affinity, as indicated by the 3 orders of magnitude increase in affinity of farnesylmethylbis(phosphonate) (28, Ki ) 6 nM) relative to farnesyl diphosphate (27, Ki ) 8 µM). The similarity of these effects suggests that the geminal phosphonates in the former may mimic the position of the pyrophosphate anion in the ion-pairing interaction demonstrated by the synergetic binding of 26a with inorganic pyrophosphate. The pimaren-8-yl ion 10+ also figures in biogenetic schemes leading to other types of tricyclic and tetracyclic diterpenes (Scheme 7).2,17,49 Migration of H9R to C8 is the first step in the sequential backbone rearrangements through 30+ which give rise to rimuene and dolabradiene. Rotation of the vinyl group and C8-C16 cyclization via 29+ presumably leads to the bridged tetracyclic diterpenes isohihabene and phyllocladene. 7004 J. AM. CHEM. SOC.

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Conclusion

The stereospecificity of the AS-catalyzed cyclization of 8Rhydroxy analogue 19a and the greater potency of pimarenylamine inhibitor 26a compared to isomers 26b,c both point to pimarenyl carbocation intermediates 10+ and 11+ having 13β methyl stereochemistry.44 A “coplanar” orientation of the copalyl diphosphate side chain and the consequent 17R-13β (17-re-13re) facial specificity in the SN′ cyclization are inferred from the contrary behavior of the epimeric 8R- and 8β-hydroxy-17nor analogues of the bicyclic intermediate. The important role of ion-pairing interactions in AS catalysis is revealed by a 2000fold enhancement of binding affinity for aza analogue 26a in the presence of pyrophosphate anion. A plausible mechanism for conversion of 9 to the abietadiene isomers 1a-4a consists of SN′ cyclization completed with vinyl group rotation and boatchair inversion, H14R f C16 si proton transfer aided by tight ion pairing with the pyrophosphate anion, 13 f 15 methyl migration, and finally proton eliminations. The resulting transformation from a sandaracopimaradiene (or sandaracopimarenyl (+)) structure to the abietadienes brings the isolated double bonds into conjugation, thus enhancing the reactivity of abietic acid and its oleoresin congeners toward atmospheric oxygen for their vital wound-sealing function in vivo. Experimental Section

Representative preparative procedures and characterization data for 8-oxo-17-norCPP (17), for norpimarenylamine 26a, and for the preparative enzymatic experiments with rAS are given below. General experimental aspects, as well as procedures and characterization data for other compounds, are available in the Supporting Information. Column chromatography was carried out as described by Still et al.50 using a 50-100 times weight excess of Merck 60 Å, grade 9385, 230-400 mesh silica gel at a pressure of 4-8 psi. The amounts, dimensions, and volumes are specified in the following format: grams of silica gel, column diameter, fraction volume, and void volume. Farnesylmethylbisphosphonate (28) was prepared according to a literature procedure.43 (48) (a) Bittner, E. W.; Arnett, E. M.; Saunders, M. J. Am. Chem. Soc. 1976, 98, 3734-3735. (b) Arnett, E. M.; Petro, C. J. Am. Chem. Soc. 1978, 100, 5408-5416. (49) Coates, R. M. In Progress in the Chemistry of Organic Natural Products; Herz, W., Grisebach, H., Kirby, J. W., Eds.; Springer-Verlag: Vienna, 1976; Vol. 33, pp 73-230. (50) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923-2925.

Mechanism of Abietadiene Synthase Catalysis

ARTICLES

(5S,9R,10S,13E)-17-Nor-8-oxo-labda-13-en-15-ol (16). Conditions for the catalytic osymylation and periodate cleavage were based on literature procedures.28,38,39 A solution of methyl copalate 13 (3.60 g, 11.3 mmol), NMO (3.44 g, 29.4 mmol), and two crystals of OsO4 (ca. 10 mg, ca. 0.039 mmol) in acetone (60 mL) was stirred for 3 h at room temperature. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (150, 44, 25, 0) using 1:1 hexanes-ethyl acetate. The yield of diols as a ca. 3:1 (based on earlier runs) mixture of regioisomers was 3.10 g (78%). A suspension of LiAlH4 (600 mg, 15.8 mmol) in ether (50 mL) was stirred and cooled at 0 °C as a solution of diols (2.29 g, 6.50 mmol) in ether (50 mL) was slowly added. The solution was allowed to warm to room temperature, and after 10 h, excess reagent was hydrolyzed by addition of water (600 µL), 15% NaOH (600 µL), and water (1.8 mL).51 The solids were filtered through Celite and washed with ether (5 × 100 mL). The combined filtrates were concentrated under reduced pressure to give a mixture of triols which was dissolved in acetone (20 mL). The solution was stirred as NaIO4 (4.15 g, 19.4 mmol) and enough water for complete dissolution were added. After 24 h, the resulting suspension was diluted with water (150 mL) and extracted with ether (3 × 100 mL). The combined ethereal extracts were dried (Na2SO4) and concentrated under reduced pressure to give a thin oil. Purification by column chromatography (80, 34, 25, 0) using 4:1 hexanes-ethyl acetate gave keto alcohol 16 (1.14 g, 60%). The bisnor methyl ketone side product arising from cleavage of the 13,14 double bond was tentatively identified by TLC comparisons with a previously prepared sample. Data for 16: 1H NMR (500 MHz, CDCl3) δ 0.69, 0.82, 0.94 (3s, 9H, 3CH3), 1.12 (td, J ) 12.6, 4.6 Hz, 1H), 1.21 (qd, J ) 13.2, 4.6 Hz, 1H), 1.2-1.4 (m, 2H), 1.42 (dtd, J ) 13.0, 3.1, 1.5 Hz, 1H), 1.46 (dd, J ) 13.0, 2.6 Hz, 1H), 1.4-1.6 (m, 2H), 1.62 (qd, J ) 13.2, 4.9 Hz, 1H), 1.64 (s, 3H, CH3), 1.71.9 (m, 3H), 1.9-2.1 (m, 3H), 2.27 (td, J ) 13.2, 7.0 Hz, 1H), 2.38 (ddd, J ) 13.2, 4.7, 2.0 Hz, 1H), 4.11 (d, J ) 7.0 Hz, 2H, CH2OH), 5.34 (t sextet, J ) 7.0, 1.3 Hz, 1H, dCH); 13C NMR (126 MHz, CDCl3) δ 14.97 (CH3), 16.41 (CH3), 19.26 (CH2), 19.84 (CH2), 21.91 (CH3), 24.35 (CH2), 33.74 (CH3), 33.94 (C), 39.13 (CH2), 39.49 (CH2), 42.18 (CH2), 42.91 (CH2), 42.94 (C), 54.54 (CH), 59.65 (CH2), 63.64 (CH), 123.78 (CH), 140.32 (C), 212.45 (C); IR (CCl4) 3622, 3483 (OH), 1710 (CdO) cm-1; [R]D20 -41.1° (c 1.27, EtOH); MS (FI) m/z 294 (M+ + 2, 7.4), 293 (M+ + 1, 24.0), 292 (M+, 100), 291 (4.8), 290 (2.6). Anal. Calcd for C19H32O2: C, 78.03; H, 11.03. Found: C, 77.65; H, 10.81. (5S,9R,10S,13E)-17-Nor-8-oxo-labda-13-en-15-yl Diphosphate, Ammonium Salt (17). Conversion of keto alcohol 16 (100 mg, 0.34 mmol) to the chloride with s-collidine (165 µL, 249 mg, 2.05 mmol), LiCl (72 mg, 1.70 mmol), and methanesulfonyl chloride (132 µL, 196 mg, 1.71 mmol) in DMF (3 mL) at 0 °C for 1.75 h followd by displacement of half of the crude chloride with tris(tetrabutylammonium) diphosphate (tribasic) (327 mg, 0.34 mmol) in acetonitrile (2 mL) for 2.3 h gave crude diphosphate 17.31,32 Purification as described for 13-aza-13,14dihydro-CPP13 by ion exchange chromatography (NH4+ form, 2.4 × 10 cm, 8-mL fractions) followed by cellulose chromatography (3 × 18 cm, 8-mL fractions) and lyophilization gave

diphosphate 17: yield 75 mg (78%); 1H NMR (400 MHz, D2O) δ 0.53, 0.67, 0.78, 1.52 (4s, 12H, 4CH3), 1.0-1.6 (m, 10H), 1.69 (dt, J ) 13.9, 7.8 Hz, 1H), 1.88 (ddd, J ) 13.7, 8.3, 6.2 Hz, 1H), 1.98 (ddt, J ) 12.7, 9.8, 2.7 Hz, 1H), 2.16 (ddd, J ) 12.9, 4.6, 2.2 Hz, 1H), 2.20 (d, J ) 13.5 Hz, 1H), 2.38 (td, J ) 12.0, 8.3 Hz, 1H), 4.29 (td, J ) 6.4, 3.9 Hz, 2H, CH2OPP), 5.22 (t, J ) 6.7 Hz, 1H, dCH); 31P NMR (162 MHz, D2O) δ -5.84, -9.72 (2d, J ) 22.0 Hz). (5S,9R,10S,13S)-15,16-Bisnor-13-isocyanatopimar-8(14)ene (25a). Conditions for the following reaction were based on those described by Yamada to prepare carbamates except that the alcohol component was omitted to isolate the isocyanate.41 A solution of acid 24a (97 mg, 0.33 mmol), triethylamine (279 µL, 203 mg, 2.01 mmol), and diphenylphosphoryl azide (225 µL, 275 mg, 1.00 mmol) in benzene (15 mL) was stirred and heated at reflux for 50 min and then cooled to room temperature. Purification by direct loading of the benzene solution onto a silica gel column (20, 24, 8, 0) followed by elution with 3% ethyl acetate in hexane provided isocyanate 25a (83 mg, 86%) as a waxy white solid: mp 50-52 °C; 1H NMR (500 MHz, CDCl3) δ 0.72, 0.82, 0.87 (3s, 9H, 3CH3), 0.99 (td, J ) 12.6, 4.4 Hz, 1H), 1.02 (dd, J ) 12.3, 2.6 Hz, 1H), 1.16 (td, J ) 13.0, 4.4 Hz, 1H), 1.27 (qd, J ) 12.8, 4.6 Hz, 1H), 1.33 (s, 3H, H13 CH3), 2.02 (tdt, J ) 13.9, 5.9, 1.7 Hz, 1H), 2.23 (ddd, J ) 14.3, 4.6, 2.0 Hz, 1H), 5.36 (q, J ) 1.3 Hz, 1H, CdCH); 13C NMR (126 MHz, CDCl3) δ 15.01 (CH3), 19.14 (CH2), 19.44 (CH2), 22.31 (CH3), 22.63 (CH2), 29.70 (CH3), 33.56 (C), 33.94 (CH2), 35.61 (CH3), 37.96 (CH2), 38.66 (C), 39.34 (CH2), 42.24 (CH2), 50.30 (CH), 54.87 (CH), 57.56 (C), 123.48 (C), 127.29 (CH), 139.78 (C); IR (CCl4) 2249 (NCO) cm-1. Anal. Calcd for C19H29NO: C, 79.39; H, 10.17; N, 4.87. Found: C, 79.21; H, 10.07; N, 4.82. (5S,9R,10S,13S)-N-Methyl-15,16-bisnorpimar-8(14)-en-13amine, Hydrochloride Salt (26a). Conditions for the following reaction were based on a review by Mu¨ller.52 Specific conditions were not given. A solution of isocyanate 25a (82 mg, 0.29 mmol) in THF (14 mL) was stirred at room temperature as solid LiAlH4 (600 mg, 15.4 mmol) was added in one portion. The solution was refluxed for 2 h until disappearance of both the starting material and the formamide intermediate was observed by TLC. Excess hydride was destroyed by addition of water (0.6 mL), 15% NaOH (0.6 mL), and water (1.8 mL)51 while cooling in an ice bath. The resulting salts were filtered out and washed with ether (100 mL). The ether filtrate was washed with 1% NaOH (100 mL), and the aqueous layer was extracted with ether (2 × 50 mL). The combined ethereal extracts were extracted with 1% HCl (4 × 50 mL) and water (1 × 25 mL), the combined aqueous layers were basified until strongly basic, and the amine product was extracted with CH2Cl2 (5 × 50 mL). The solution was dried (Na2SO4) and concentrated under reduced pressure to a white solid (75 mg). The crude amine in ether (1 mL) was converted to the hydrochloride salt with 1 M HCl in ether (0.4 mL). Recrystallization twice from absolute ethanol (